In order to understand how a pathogenic mutation causes inherited eye disease, it is necessary recognize how pathogenic mutations could affect protein structure-function, metabolic pathways, and how these perturbations could be associated clinical parameters describing the disease phenotype. For this purpose we perform molecular modeling to build protein structure, evaluate the severity of genetic missense changes from the atomic level of protein, and provide a quantitative analysis of the mutation impact on protein structure, stability and catalytic activity. We also do experimental in vitro studies for proteins of interest to measure the protein fold destabilization and changes in catalytic activity caused by the disease-related mutations. Finally we correlate these findings with clinical phenotypes from inherited eye disease. In addition, in collaboration with the National Center for Advancing Translational Sciences (NCATS) we search for drug activators of catalytic activity of mutant protein affected by genetic mutation. This year we were using oculocutaneous albinism, Stargardts macular degeneration, choroideremia (CHM), Leber congenital amaurosis (LCA), and others as our disease models. Oculocutaneous albinism is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with no or reduced TYR activity are classified as OCA1A or OCA1B forms, respectively. This year we performed large scale protein purifications for the human tyrosinase. In total 70 mg of catalytically active tyrosinase intra-melanosomal domain (truncated tyrosinase) were purified from the larval biomass and were a subject of high-throughput screens at the NCATS. To date 17,011 compounds from the Genesis Drug Collection, 2,108 compounds from the Natural Products Library, and 2,816 compounds from the NCATS Pharmaceutical Collection were successfully screened. First search has found new inhibitors (>100) and several activators of tyrosinase. Recently we also purified a recombinant full-length tyrosinase, which contains both, intra-melanosomal and trans-membrane domains. We demonstrated that full-length and truncated tyrosinases have similar enzymatic activities. This study validates our drug screening, where we are using the truncated protein. In addition, we performed biochemical, biophysical, and in silico studies of human recombinant tyrosinases to understand folding and stability of missense changes R402Q, P406L, R422Q, and R422W mimicking mutations in OCA1B form of albinism. We also implied molecular modeling to investigate the potential structural and functional consequences as well as possible risks associated with genetic mutations causing inherited eye diseases. Autosomal recessive Stargardts disease is the most common form of juvenile macular dystrophy and results from mutations in the ABCA4 gene. Around 50% of pathogenic ABCA4 missense mutations occur in the trans-membrane or nucleotide binding domains. The atomic structure of these domains obtained using molecular modeling. We improved molecular modeling of disease causing mutations and testing our predictions with clinical data, which were significantly expanded on this year. CHM is an X-linked degeneration of the retinal pigment epithelium, photoreceptors, and choroid, which causes nyctalopia and progressive constriction of visual fields leading to blindness. The CHM gene encodes Rab escort protein 1 (REP-1). We performed molecular modeling of a REP-1 functional protein complex (Freund P.R., Sergeev Y.V., MacDonald I.M., Molecular Genetics & Genomics Medicine, 2016). A retrospective review of 128 affected males was performed analyzing the onset of symptoms, visual acuity, and visual fields with respect to their mutations in the CHM gene. In the pool of 106 CHM mutations, four novel missense mutations were discovered. The mutations, L80F, Q273H, M443V, and L457P, predicted to be severe changes affecting protein stability and folding with the effect similar to that of other types of mutations currently known for CHM. Therefore, all CHM patients have the Loss of Function mutations and show no functional REP-1. This result confirms the clinical data shown no significant genotype-phenotype correlation in respect to the onset of nyctalopia, the onset of other visual symptoms, visual acuity, or width of visual fields. We also modeled the structure for GUCY2 protein to show a role of genetic mutation in LCA (Gradstein L. et al., BMC Medical Genetics, 2016). The purpose of this study was to clinically characterize and identify the cause of disease in a large inbred Bedouin Israeli tribe with LCA. Sequencing of GUCY2D identified a novel missense mutation (c.2129C>T; p.Ala710Val) resulting in substitution of alanine by valine at position 710 within the protein kinase domain of the retina-specific enzyme guanylate cyclase 1 (GC1) encoded by GUCY2D. Molecular modeling implied that the mutation changes the conformation of the regulatory segment within the kinase styk-domain of GC1 and causes loss of its helical structure, likely inhibiting phosphorylation of threonine residue within this segment, which is needed to activate the catalytic domain of the protein. This is the first documentation of the p.Ala710Val mutation in GC1 and the second ever described mutation in its protein kinase domain. These findings enlarge the scope of genetic variability of LCA, highlight the phenotypic heterogeneity found amongst individuals harboring an identical LCA mutation, and possibly provide hope for gene therapy in patients with this congenital blinding disease. As the Bedouin kindred studied originates from Saudi Arabia, the mutation found might be an ancient founder mutation in that large community. Another target for genetic mutations in LCA is molecular chaperone CCT2 (Minegishi et al., Scientific Reports-Nature, 2016). To predict whether these mutants have actual impacts on disease onset, molecular modeling was conducted. Atomic structure of each of 8 domains was individually modeled by homology. The final structure of hetero-octamer was built using the bovine TRIC/CCT chaperone as a structural template. The CCT2 missense mutations T400P and R516H were generated in optimized conformation using the 1ns molecular dynamics in water. The change of the proline residue in position 400 disrupt alpha-helical conformation and destabilizes several hydrogen bonds in the area of C-cap of the helix 14. In addition, the introduction of relatively bulky proline residue in the interface between alpha-helices could cause outward movement of surrounding helices. This movement could potentially reduce the nucleotide binding maintained by these helices. Another mutant, R516H, is located in the C-cap of alpha-helix 18. In CCT2 subunit positively charged arginine residue (R516) additionally stabilize alpha-helix 18 by forming a salt bridge with negatively charged glutamic acid E509. Mutation to histidine residue breaks the salt bridge that predicted to loosen the alpha-helix structure. It is known that intermediate alpha-helices yielding these mutations are important for CCT-chaperonin intra-ring formation. Identified unrivalled candidate CCT2 and its mutants T400P and R516H proteins were predicted to have structural decays and indeed were biochemically instable.